Methods and compositions for improving skeletal muscle protein fractional synthetic rate

ABSTRACT

Methods and egg yolk powder containing compositions for improving skeletal muscle protein fractional synthetic rate (FSR) in mammals and inhibiting or reversing sarcopenia are provided.

This patent application is a divisional of U.S. patent application Ser.No. 16/508,814, filed Jul. 11, 2019, which claims the benefit ofpriority from U.S. Provisional Application Ser. No. 62/756,286 filedNov. 6, 2018, the teachings of each of which are incorporated byreference in their entireties.

FIELD

Embodiments of the present invention are generally related to methodsand compositions for improving skeletal muscle protein fractionalsynthetic rate (FSR) in mammals.

BACKGROUND

Muscle loss represents one of the most important causes of functionaldecline and loss of independence in older adults. Age-related muscleloss or sarcopenia is commonly observed in many older adults and is alsocharacterized by a decrease in the rate of muscle protein synthesis. Inaddition, low muscle mass is associated with fall-related injuries whichcan be devastating to adults over the age of 60 years, particularly ifthey result in bone fractures.

Myostatin, first described in 1997, is a protein in the transforminggrowth factor β superfamily that is produced by myotubes and can act inan endocrine, autocrine, and paracrine fashion (McPherron et al. Nature1997 387(6628):83-90). Myostatin binds to a transmembrane receptorprotein on myocytes, activin receptor type IIB, to inhibit the Akt-mTORpathway, upregulate activity of the ubiquitin-proteasome pathway, andprevent synthesis of satellite cell replication and translocation(myoblast proliferation). Activation of these various pathways eitherinhibits muscle hypertrophy or promotes muscle atrophy. Inhibition ofmyostatin by various methods has been shown to interrupt these variouspathways, reversing or preventing muscle atrophy in various rodentmodels of disease states, including disuse atrophy (Bogdanovich et al.Nature 2012 420(6914): 418; Holzbaur et al. Neurobiology of disease 200623.3: 697-707; Klimek et al. Biochemical and biophysical researchcommunications 2010 391(3):1548-1554; Murphy et al. Journal of appliedphysiology 2011 110(4):1065-1072). Long-term myostatin inhibition bygene transfer therapy resulted in increased muscle mass in goldenretrievers with muscular dystrophy (Bish et al. Human gene therapy 201122(12): 1499-1509).

Recommendations for preventing sarcopenia include exercise, inparticular resistance training and adequate nutrition inclusive ofprotein balanced with non-acid producing foods fruits and vegetables.See https:// with the extension iofbonehealth.org/treating-sarcopenia ofthe world wide web.

Creatine supplements have been indicated to aid in muscle developmentfor older adults that are following a resistance training program (Broseet al. J Gerontol A Biol Sci Med Sci. 2003 58(1):11-9; Chrusch et al.Med Sci Sports Exerc. 2001 33(12):2111-7).

Maintaining appropriate blood levels of vitamin D may also aid inmaintaining muscle strength and physical performance (Mithal et al.Osteoporos Int. 2013 May; 24(5):1555-66).

While high-dose testosterone increases muscle power and function, buthas a number of potentially limiting side effects (Morley, J. E. CalcifTissue Int. 2016 April; 98(4):319-33).

Other drugs in clinical development include selective androgen receptormolecules, ghrelin agonists, myostatin antibodies, activin IIRantagonists, angiotensin converting enzyme inhibitors, beta antagonists,and fast skeletal muscle troponin activators (Morley, J. E. CalcifTissue Int. 2016 April; 98(4):319-33).

As sarcopenia is a major predictor of frailty, hip fracture, disability,and mortality in older adults (Morley, J. E. Calcif Tissue Int. 2016April; 98(4):319-33), agents which inhibit and/or reverse sarcopenia areneeded.

SUMMARY

An aspect of the present invention relates to a method for improvingskeletal muscle protein fractional synthetic rate (FSR) in mammals. Themethod comprises administering to the mammal a composition comprisingegg yolk powder.

In one nonlimiting embodiment, the mammal is an older adult.

In one nonlimiting embodiment, the composition administered isFORTETROPIN.

Another aspect of the present invention relates to a method forinhibiting or reversing sarcopenia in a mammal. The method comprisesadministering to the mammal a composition comprising egg yolk powder.

In one nonlimiting embodiment, the mammal is an older adult.

In one nonlimiting embodiment, the composition administered isFORTETROPIN.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a diagram of the design of a study showing the effectsof FORTETROPIN on skeletal muscle protein FSR.

FIG. 2 shows results comparing myofibril proteins fractional synthesisin older adults receiving FORTETROPIN versus cheese powder for 21 days.

FIG. 3 shows results comparing cytoplasmic proteins fractional synthesisin older adults receiving FORTETROPIN versus cheese powder for 21 days.

FIG. 4 shows results comparing mitochondrial proteins fractionalsynthesis in older adults receiving FORTETROPIN versus cheese powder for21 days.

FIG. 5 shows results comparing membrane proteins fractional synthesis inolder adults receiving FORTETROPIN versus cheese powder for 21 days.

DETAILED DESCRIPTION

The headings used herein are for organizational purposes only and arenot meant to be used to limit the scope of the description or theclaims. As used throughout this application, the word “may” is used in apermissive sense (i.e., meaning having the potential to), rather thanthe mandatory sense (i.e., meaning must). Similarly, the words“include”, “including”, and “includes” mean including but not limitedto. To facilitate understanding, like reference numerals have been used,where possible, to designate like elements common to the figures.

The present invention provides methods and compositions for improvingskeletal muscle protein fractional synthetic rate (FSR) in mammals. Themethods comprise administering to the mammal a composition comprisingegg yolk powder.

For purposes of the present invention, by the terms “improve”,“improving” or “improvement” with respect to skeletal muscle proteinfractional synthetic rate, it is meant an increase in synthesis ofskeletal muscle proteins indicative of muscle growth.

Methods and compositions of the present invention are useful ininhibiting and/or reversing sarcopenia in mammals.

Compositions administered in accordance with the present inventioncomprise egg yolk powder or one or more proteins and/or lipids derivedfrom egg yolk which are effective in growth and development of leanmuscle tissue.

In one nonlimiting embodiment, the composition administered isFORTETROPIN. FORTETROPIN is a fertilized egg yolk derived product usedas a dietary and nutritional supplement (MYOS RENS TECHNOLOGY INC.CORPORATION Cedar Knolls, N.J.). FORTETROPIN has been found to have anabundance of pro-angiogenic and host-defense proteins when compared tounfertilized egg yolk. When administered to humans and rats, FORTETROPINhas been shown to reduce serum myostatin levels and demonstrate a changein downstream signaling pathways supportive of muscle hypertrophy andresulting in an increase in lean body mass. FORTETROPIN may be producedvia high pressure pasteurization (HPP) and freeze drying of at least aportion of an egg. A method for production of FORTETROPIN is disclosedin U.S. Pat. No. 8,815,320, teachings of which are herein incorporatedby reference in their entirety.

In one nonlimiting embodiment, FORTETROPIN production is optimized toenhance potency as it relates to muscle disuse atrophy by modifying oneor more egg yolk-related parameters such as, but not limited to,incubation time post-lay, fertility status and breed of chicken.

In another nonlimiting embodiment, the composition comprises an avianfollistatin such as described in U.S. Published Patent Application No.2007/0275036, the disclosure of which is incorporated herein byreference in its entirety and/or other proteins and/or lipids found inavian eggs and which are beneficial in growth and development of leanmuscle tissue such as, but not limited to, those described in U.S.Provisional Application Ser. No. 62/755,601, the disclosure of which isincorporated herein by reference in its entirety.

For purposes of the present invention, when the phrases “egg yolkpowder” or “composition comprising egg yolk powder” are used, they aremeant to be inclusive, but are not limited to, egg yolk powder, one ormore proteins and/or lipids derived from egg yolk which are effective ingrowth and development of lean muscle tissue, FORTETROPIN and avianfollistatin.

By “mammal” or “mammals” it is meant to include, but is not limited to,humans, apes, monkeys, cows, sheep, goats, dogs, cats, mice, rats, andtransgenic species thereof.

In one nonlimiting embodiment, the mammal is an older adult. In onenonlimiting embodiment, the older adult is age 60 or greater.

In one nonlimiting embodiment, the composition comprising egg yolkpowder is administered orally on a daily basis, one, two or three timesa day.

In one nonlimiting embodiment, the composition comprising egg yolkpowder is administered in an amount effective to upregulate mTor pathwayactivity, downregulate ubiquitin proteasome pathway activity,downregulate serum myostatin levels and/or reduce ActRIIB expression, areceptor for myostatin.

In one nonlimiting embodiment, the composition comprising egg yolkpowder is FORTETROPIN administered orally on a daily basis in an amountranging from about 5 to about 25 grams/day, about 6.6 to about 19.8grams/day or about 50 to about 300 mg/kg/day, about 80 to about 250mg/kg/day in humans. In canines, FORTETROPIN is administered orally on adaily basis in an amount ranging from about 200-1000 mg/kg/day, about300 to about 900 mg/kg/day.

In some embodiments, the composition comprising egg yolk powder isadministered at 300 mg/kg daily (one scoop (6600 mg)/22 kg), or the likeor a suitable dosage for the weight and characteristics of the mammal.Dosages may be modified for efficacy, for example, may be administeredat a higher or lower dosage or administered more than once daily or lessthan once daily. Mammals may be dosed to the closest ½ scoop, or thelike, without underdosing. The composition may be formed in a powderthat may be mixed with other food to facilitate ingestion.

However, as will be understood by the skilled artisan upon reading thisdisclosure, the compositions described herein can be formulated foradministration to a subject via any conventional means including, butnot limited to, oral, or buccal.

Moreover, the compositions described herein, can be formulated into anysuitable dosage form, including but not limited to, aqueous oraldispersions, liquids, gels, syrups, elixirs, slurries, suspensions andthe like, for oral ingestion by an individual in need, solid oral dosageforms, controlled release formulations, fast melt formulations,effervescent formulations, lyophilized formulations, tablets, powders,pills, dragees, capsules, delayed release formulations, aqueous liquiddispersions, self-emulsifying dispersions, solid solutions, liposomaldispersions, solid dosage forms, powders, tablets, capsules, pills,delayed release formulations.

Formulations for oral use can be obtained by mixing one or more solidexcipient with one or more of the compounds described herein, optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients include, for example, fillers such assugars, including glucose, fructose, lactose, sucrose, mannitol,sorbitol, stevia extract, or sucralose; cellulose preparations such as,for example, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose,hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or otherssuch as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. Ifdesired, disintegrating agents may be added, such as the cross-linkedcroscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or asalt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Formulations which can be used orally include push-fit capsules made ofgelatin, as well as soft, sealed capsules made of gelatin and aplasticizer, such as glycerol or sorbitol. The push-fit capsules cancontain the active ingredients in admixture with fillers such aslactose, binders such as starches, and/or lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive compounds may be dissolved or suspended in suitable liquids, suchas fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for such administration.

In some embodiments, the solid dosage forms disclosed herein may be inthe form of a tablet, (including a suspension tablet, a fast-melttablet, a bite-disintegration tablet, a rapid-disintegration tablet, aneffervescent tablet, or a caplet), a pill, a powder (including a sterilepackaged powder, a dispensable powder, or an effervescent powder) acapsule (including both soft or hard capsules, e.g., capsules made fromanimal-derived gelatin or plant-derived HPMC, or “sprinkle capsules”),solid dispersion, solid solution, pellets, granules. In otherembodiments, the pharmaceutical formulation is in the form of a powder.In still other embodiments, the pharmaceutical formulation is in theform of a tablet. Additionally, formulations described herein may beadministered as a single capsule or in multiple capsule dosage form. Insome embodiments, the formulation is administered in two, or three, orfour, capsules or tablets.

Soft gel or soft gelatin capsules may be prepared, for example, withoutlimitation, by dispersing the formulation in an appropriate vehicle(vegetable oils are commonly used) to form a high viscosity mixture.This mixture is then encapsulated with a gelatin-based film usingtechnology and machinery known to those in the soft gel industry. Theindustrial units so formed are then dried to constant weight.

In some embodiments, the formulations may include other medicinal orpharmaceutical agents, carriers, diluents, dispersing agents, suspendingagents, thickening agents, adjuvants, such as preserving, stabilizing,wetting or emulsifying agents, solution promoters, and/or buffers. Inaddition, the formulations can also contain other therapeuticallyvaluable substances.

The formulations described herein can include egg yolk powder and one ormore pharmaceutically acceptable additives such as a compatible carrier,binder, filling agent, suspending agent, flavoring agent, sweeteningagent, disintegrating agent, dispersing agent, surfactant, lubricant,colorant, diluent, solubilizer, moistening agent, plasticizer,stabilizer, penetration enhancer, wetting agent, anti-foaming agent,antioxidant, preservative, or one or more combination(s) thereof. Instill other aspects, using standard coating procedures, a film coatingis provided around the formulation of the compound described herein. Inone embodiment, some or all of the particles of the compound describedherein are coated. In another embodiment, some or all of the particlesof the compound described herein are microencapsulated. In still anotherembodiment, the particles of the compound described herein are notmicroencapsulated and are uncoated.

In certain embodiments, compositions may also include one or more pHadjusting agents or buffering agents, including acids such as acetic,boric, citric, lactic, phosphoric and hydrochloric acids; bases such assodium hydroxide, sodium phosphate, sodium borate, sodium citrate,sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; andbuffers such as citrate/dextrose, sodium bicarbonate and ammoniumchloride. Such acids, bases and buffers are included in an amountrequired to maintain pH of the composition in an acceptable range.

In other embodiments, compositions may also include one or more salts inan amount required to bring osmolality of the composition into anacceptable range. Such salts include those having sodium, potassium orammonium cations and chloride, citrate, ascorbate, borate, phosphate,bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable saltsinclude sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

Formulations including egg yolk powder, as described herein, may bemanufactured in a conventional manner, such as, by way of example only,by means of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping or compressionprocesses.

In certain embodiments, compositions provided herein may also includeone or more preservatives to inhibit microbial activity. Suitablepreservatives include mercury-containing substances such as merfen andthiomersal; stabilized chlorine dioxide; and quaternary ammoniumcompounds such as benzalkonium chloride, cetyltrimethylammonium bromideand cetylpyridinium chloride.

Formulations described herein may benefit from antioxidants, metalchelating agents, thiol containing compounds and other generalstabilizing agents. Examples of such stabilizing agents, include, butare not limited to: (a) about 0.5% to about 2% w/v glycerol, (b) about0.1% to about 1% w/v methionine, (c) about 0.1% to about 2% w/vmonothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% toabout 2% w/v ascorbic acid, (f) 0.003% to about 0.02% w/v polysorbate80, (g) 0.001% to about 0.05% w/v. polysorbate 20, (h) arginine, (i)heparin, (j) dextran sulfate, (k) cyclodextrins, (l) pentosanpolysulfate and other heparinoids, (m) divalent cations such asmagnesium and zinc; or (n) combinations thereof.

Binders imparting cohesive qualities may also be used. Examples include,but are not limited to, alginic acid and salts thereof; cellulosederivatives such as carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, ethylcellulose, and microcrystalline cellulose;microcrystalline dextrose; amylose; magnesium aluminum silicate;polysaccharide acids; bentonites; gelatin; polyvinylpyrrolidone/vinylacetate copolymer; crosspovidone; povidone; starch; pregelatinizedstarch; tragacanth, dextrin, a sugar, such as sucrose, glucose,dextrose, molasses, mannitol, sorbitol, xylitol, and lactose; a naturalor synthetic gum such as acacia, tragacanth, ghatti gum, mucilage ofisapol husks, polyvinylpyrrolidone, larch arabogalactan, polyethyleneglycol, waxes, sodium alginate, and the like.

In general, binder levels of 20-70% are used in powder-filled gelatincapsule formulations. Binder usage level in tablet formulations varieswhether direct compression, wet granulation, roller compaction, or usageof other excipients such as fillers which itself can act as moderatebinder.

Formulators skilled in art can determine the binder level for theformulations, but binder usage level of up to 70% in tablet formulationsis common.

Compositions may further comprise carriers of relatively nontoxicchemical compounds or agents that facilitate the incorporation of acompound into cells or tissues. Nonlimiting examples include binders,suspending agents, disintegration agents, filling agents, surfactants,solubilizers, stabilizers, lubricants, wetting agents, diluents, and thelike. Suitable carriers for use in solid dosage forms described hereininclude, but are not limited to, acacia, gelatin, colloidal silicondioxide, calcium glycerophosphate, calcium lactate, maltodextrin,glycerine, magnesium silicate, sodium caseinate, soy lecithin, sodiumchloride, tricalcium phosphate, dipotassium phosphate, sodium stearoyllactylate, carrageenan, monoglyceride, diglyceride, pregelatinizedstarch, hydroxypropylmethylcellulose, hydroxypropylmethylcelluloseacetate stearate, sucrose, microcrystalline cellulose, lactose, mannitoland the like.

Dispersing agents and/or viscosity modulating agents include materialsthat control the diffusion and homogeneity of a compound through liquidmedia or a granulation method or blend method. In some embodiments,these agents also facilitate the effectiveness of a coating or erodingmatrix. Nonlimiting examples of diffusion facilitators/dispersing agentsinclude hydrophilic polymers, electrolytes, a Tween, PEG,polyvinylpyrrolidone, and carbohydrate-based dispersing agents such ashydroxypropyl celluloses (e.g., HPC, HPC-SL, and HPC-L), hydroxypropylmethylcelluloses (e.g., HPMC K100, HPMC K4M, HPMC K15M, and HPMC K100M),carboxymethylcellulose sodium, methylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropylmethylcellulose phthalate,hydroxypropylmethylcellulose acetate stearate (HPMCAS), noncrystallinecellulose, magnesium aluminum silicate, triethanolamine, polyvinylalcohol (PVA), vinyl pyrrolidone/vinyl acetate copolymer (S630),4-(1,1,3,3-tetramethylbutyl)-phenol polymer with ethylene oxide andformaldehyde (also known as tyloxapol), poloxamers, block copolymers ofethylene oxide and propylene oxide; and poloxamines, tetrafunctionalblock copolymers derived from sequential addition of propylene oxide andethylene oxide to ethylenediamine, polyvinylpyrrolidone K12,polyvinylpyrrolidone K17, polyvinylpyrrolidone K25, orpolyvinylpyrrolidone K30, polyvinylpyrrolidone/vinyl acetate copolymer(S-630), polyethylene glycol, e.g., the polyethylene glycol can have amolecular weight of about 300 to about 6000, or about 3350 to about4000, or about 7000 to about 5400, sodium carboxymethylcellulose,methylcellulose, polysorbate-80, sodium alginate, gums, such as, e.g.,gum tragacanth and gum acacia, guar gum, xanthans, including xanthangum, sugars, cellulosics, such as, e.g., sodium carboxymethylcellulose,methylcellulose, sodium carboxymethylcellulose, polysorbate-80, sodiumalginate, polyethoxylated sorbitan monolaurate, polyethoxylated sorbitanmonolaurate, povidone, carbomers, polyvinyl alcohol (PVA), alginates,chitosans and combinations thereof. Plasticizers such as cellulose ortriethyl cellulose can also be used as dispersing agents. Dispersingagents that are particularly useful in liposomal dispersions andself-emulsifying dispersions are dimyristoyl phosphatidyl choline,natural phosphatidyl choline from eggs, natural phosphatidyl glycerolfrom eggs, cholesterol and isopropyl myristate.

Combinations of one or more erosion facilitator with one or morediffusion facilitator can also be used in the present compositions.

Compositions of the present invention may further comprise diluents usedto dilute the compound of interest prior to delivery. Diluents can alsobe used to stabilize compounds because they can provide a more stableenvironment. Salts dissolved in buffered solutions (which also canprovide pH control or maintenance) are utilized as diluents in the art,including, but not limited to a phosphate buffered saline solution. Incertain embodiments, diluents increase bulk of the composition tofacilitate compression or create sufficient bulk for homogenous blendfor capsule filling. Such compounds include e.g., lactose, starch,mannitol, sorbitol, dextrose, microcrystalline cellulose; dibasiccalcium phosphate, dicalcium phosphate dihydrate; tricalcium phosphate,calcium phosphate; anhydrous lactose, spray-dried lactose;pregelatinized starch, compressible sugar; mannitol,hydroxypropylmethylcellulose, hydroxypropylmethylcellulose acetatestearate, sucrose-based diluents, confectioner's sugar; monobasiccalcium sulfate monohydrate, calcium sulfate dihydrate; calcium lactatetrihydrate, dextrates; hydrolyzed cereal solids, amylose; powderedcellulose, calcium carbonate; glycine, kaolin; sodium chloride;inositol, bentonite, and the like.

Compositions may further comprise an enteric coating, a substance thatremains substantially intact in the stomach but dissolves and releasesthe egg yolk powder in the small intestine or colon. Generally, theenteric coating comprises a polymeric material that prevents release inthe low pH environment of the stomach but that ionizes at a higher pH,typically a pH of 6 to 7, and thus dissolves sufficiently in the smallintestine or colon to release the active agent therein.

In addition, the compositions may comprise an erosion facilitator, amaterial that controls the erosion of a particular material ingastrointestinal fluid. Erosion facilitators are generally known tothose of ordinary skill in the art. Exemplary erosion facilitatorsinclude, e.g., hydrophilic polymers, electrolytes, proteins, peptides,and amino acids.

Filling agents including compounds such as lactose, calcium carbonate,calcium phosphate, dibasic calcium phosphate, calcium sulfate,microcrystalline cellulose, cellulose powder, dextrose, dextrates,dextran, starches, pregelatinized starch, sucrose, xylitol, lactitol,mannitol, sorbitol, sodium chloride, polyethylene glycol, and the likecan also be included in the compositions. Suitable filling agents foruse in the solid dosage forms described herein include, but are notlimited to, lactose, calcium carbonate, calcium phosphate, dibasiccalcium phosphate, calcium sulfate, microcrystalline cellulose,cellulose powder, dextrose, dextrates, dextran, starches, pregelatinizedstarch, hydroxypropylmethycellulose (HPMC), hydroxypropylmethycellulosephthalate, hydroxypropylmethylcellulose acetate stearate (HPMCAS),sucrose, xylitol, lactitol, mannitol, sorbitol, sodium chloride,polyethylene glycol, and the like.

In addition, flavoring agents and/or sweeteners can be used in thecompositions and may include acacia syrup, acesulfame K, alitame, anise,apple, aspartame, banana, Bavarian cream, berry, black currant,butterscotch, calcium citrate, camphor, caramel, cherry, cherry cream,chocolate, cinnamon, bubble gum, citrus, citrus punch, citrus cream,cotton candy, cocoa, cola, cool cherry, cool citrus, cyclamate,cylamate, dextrose, eucalyptus, eugenol, fructose, fruit punch, ginger,glycyrrhetinate, glycyrrhiza (licorice) syrup, grape, grapefruit, honey,isomalt, lemon, lime, lemon cream, monoammonium glyrrhizinate, maltol,mannitol, maple, marshmallow, menthol, mint cream, mixed berry,neohesperidine DC, neotame, orange, pear, peach, peppermint, peppermintcream, raspberry, root beer, rum, saccharin, safrole, sorbitol,spearmint, spearmint cream, strawberry, strawberry cream, stevia,sucralose, sucrose, sodium saccharin, saccharin, aspartame, acesulfamepotassium, mannitol, talin, sylitol, sucralose, sorbitol, Swiss cream,tagatose, tangerine, thaumatin, tutti fruitti, vanilla, walnut,watermelon, wild cherry, wintergreen, xylitol, or any combination ofthese flavoring ingredients, e.g., anise-menthol, cherry-anise,cinnamon-orange, cherry-cinnamon, chocolate-mint, honey-lemon,lemon-lime, lemon-mint, menthol-eucalyptus, orange-cream, vanilla-mint,and mixtures thereof.

The compositions may further comprise lubricants and/or glidants thatprevent, reduce or inhibit adhesion or friction of materials.Nonlimiting examples of lubricants include stearic acid, calciumhydroxide, talc, sodium stearyl fumerate, a hydrocarbon such as mineraloil, or hydrogenated vegetable oil such as hydrogenated soybean oil,higher fatty acids and their alkali-metal and alkaline earth metalsalts, such as aluminum, calcium, magnesium, zinc, stearic acid, sodiumstearates, glycerol, talc, waxes, boric acid, sodium benzoate, sodiumacetate, sodium chloride, leucine, a polyethylene glycol (e.g.,PEG-4000) or a methoxypolyethylene glycol, sodium oleate, sodiumbenzoate, glyceryl behenate, polyethylene glycol, magnesium or sodiumlauryl sulfate, colloidal silica, a starch such as corn starch, siliconeoil, a surfactant, and the like.

Plasticizers, compounds used to soften the microencapsulation materialor film coatings to make them less brittle may also be included in thecompositions. Examples of suitable plasticizers include, but are notlimited to, polyethylene glycols such as PEG 300, PEG 400, PEG 600, PEG1450, PEG 3350, and PEG 800, stearic acid, propylene glycol, oleic acid,triethyl cellulose and triacetin. In some embodiments, plasticizers canalso function as dispersing agents or wetting agents.

The compositions may further comprise solubilizers such as triacetin,triethylcitrate, ethyl oleate, ethyl caprylate, sodium lauryl sulfate,sodium doccusate, vitamin E TPGS, dimethylacetamide,N-methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone,hydroxypropylmethyl cellulose, hydroxypropyl cyclodextrins, ethanol,n-butanol, isopropyl alcohol, cholesterol, bile salts, polyethyleneglycol 200-600, glycofurol, transcutol, propylene glycol, and dimethylisosorbide and the like.

In addition, the compositions my comprise stabilizers such asantioxidation agents, buffers, acids, preservatives and the like.

Suitable suspending agents for use in solid dosage forms described hereinclude, but are not limited to, polyvinylpyrrolidone, e.g.,polyvinylpyrrolidone K12, polyvinylpyrrolidone K17, polyvinylpyrrolidoneK25, or polyvinylpyrrolidone K30, polyethylene glycol, e.g., thepolyethylene glycol can have a molecular weight of about 300 to about6000, or about 3350 to about 4000, or about 7000 to about 5400, vinylpyrrolidone/vinyl acetate copolymer (S630), sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,polysorbate-80, hydroxyethylcellulose, sodium alginate, gums, such as,e.g., gum tragacanth and gum acacia, guar gum, xanthans, includingxanthan gum, sugars, cellulosics, such as, e.g., sodiumcarboxymethylcellulose, methylcellulose, sodium carboxymethylcellulose,hydroxypropylmethylcellulose, hydroxyethylcellulose, polysorbate-80,sodium alginate, polyethoxylated sorbitan monolaurate, polyethoxylatedsorbitan monolaurate, povidone and the like.

Surfactants including compounds such as sodium lauryl sulfate, sodiumdocusate, Tweens, triacetin, vitamin E TPGS, sorbitan monooleate,polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bilesalts, glyceryl monostearate, copolymers of ethylene oxide and propyleneoxide and the like may also be included. Additional surfactants includepolyoxyethylene fatty acid glycerides and vegetable oils, e.g.,polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylenealkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Insome embodiments, surfactants may be included to enhance physicalstability or for other purposes.

Viscosity enhancing agents including, e.g., methyl cellulose, xanthangum, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetatestearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinylalcohol, alginates, acacia, chitosans and combinations thereof may alsobe included.

In addition, wetting agents including compounds such as oleic acid,glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate,triethanolamine oleate, polyoxyethylene sorbitan monooleate,polyoxyethylene sorbitan monolaurate, sodium docusate, sodium oleate,sodium lauryl sulfate, sodium doccusate, triacetin, Tween 80, vitamin ETPGS, ammonium salts and the like may be included in these compositions.

In some embodiments, solid dosage forms, e.g., tablets, capsules, areprepared by mixing the egg yolk powder described herein, with one ormore pharmaceutical excipients to form a bulk blend composition. Whenreferring to these bulk blend compositions as homogeneous, it is meantthat the particles of egg yolk powder, are dispersed evenly throughoutthe composition so that the composition may be readily subdivided intoequally effective unit dosage forms, such as tablets, pills, andcapsules.

Conventional techniques include, e.g., one or a combination of methods:(1) dry mixing, (2) direct compression, (3) milling, (4) dry ornon-aqueous granulation, (5) wet granulation, or (6) fusion. See, e.g.,Lachman et al., “The Theory and Practice of Industrial Pharmacy” (1986).

It should be appreciated that there is considerable overlap betweenadditives used in the solid dosage forms described herein. Thus, theabove-listed additives should be taken as merely exemplary, and notlimiting, of the types of additives that can be included.

A capsule may be prepared, for example, by placing the bulk blend of theformulation of the compound described above, inside of a capsule. Insome embodiments, the formulations (non-aqueous suspensions andsolutions) are placed in a soft gelatin capsule. In other embodiments,the formulations are placed in standard gelatin capsules or non-gelatincapsules such as capsules comprising HPMC. In other embodiments, theformulation is placed in a sprinkle capsule, wherein the capsule may beswallowed whole or the capsule may be opened and the contents sprinkledon food prior to eating. In some embodiments, the therapeutic dose issplit into multiple (e.g., two, three, or four) capsules. In someembodiments, the entire dose of the formulation is delivered in acapsule form.

In another aspect, dosage forms may include microencapsulatedformulations. In some embodiments, one or more other compatiblematerials are present in the microencapsulation material. Exemplarymaterials include, but are not limited to, pH modifiers, erosionfacilitators, anti-foaming agents, antioxidants, flavoring agents, andcarrier materials such as binders, suspending agents, disintegrationagents, filling agents, surfactants, solubilizers, stabilizers,lubricants, wetting agents, and diluents.

Materials useful for the microencapsulation described herein includematerials which sufficiently isolate the compound from othernon-compatible excipients. Materials compatible with the egg yolk powderare those that delay the release of the egg yolk powder in vivo.

In other embodiments, the formulations described herein, which includethe egg yolk powder, are solid dispersions. Methods of producing suchsolid dispersions are known in the art and include, but are not limitedto, for example, U.S. Pat. Nos. 4,343,789, 5,340,591, 5,456,923,5,700,485, 5,723,269, and U.S. Pub. Appl 2004/0013734.

In still other embodiments, the formulations described herein are solidsolutions. Solid solutions incorporate a substance together with theactive agent and other excipients such that heating the mixture resultsin dissolution of the drug and the resulting composition is then cooledto provide a solid blend which can be further formulated or directlyadded to a capsule or compressed into a tablet. Methods of producingsuch solid solutions are known in the art and include, but are notlimited to, for example, U.S. Pat. Nos. 4,151,273, 5,281,420, and6,083,518.

In some embodiments, the solid dosage forms described herein can beformulated as enteric coated delayed release oral dosage forms, i.e., asan oral dosage form of a pharmaceutical composition as described hereinwhich utilizes an enteric coating to affect release in the smallintestine of the gastrointestinal tract. The enteric coated dosage formmay be a compressed or molded or extruded tablet/mold (coated oruncoated) containing granules, powder, pellets, beads or particles ofthe active ingredient and/or other composition components. The entericcoated oral dosage form may also be a capsule (coated or uncoated)containing pellets, beads or granules of the solid carrier or thecomposition.

The term “delayed release” as used herein refers to the delivery so thatthe release can be accomplished at some generally predictable locationin the intestinal tract more distal to that which would have beenaccomplished if there had been no delayed release alterations. In someembodiments the method for delay of release is coating. Any coatingsshould be applied to a sufficient thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below about 5,but does dissolve at pH about 5 and above. It is expected that anyanionic polymer exhibiting a pH-dependent solubility profile can be usedas an enteric coating for the methods and compositions described hereinto achieve delivery to the lower gastrointestinal tract.

In some embodiments, formulations are provided that include particles ofegg yolk powder described herein and at least one dispersing agent orsuspending agent for oral administration to a subject. The formulationsmay be a powder and/or granules for suspension, and upon admixture withwater, a substantially uniform suspension is obtained. Liquidformulation dosage forms for oral administration can be aqueoussuspensions selected from the group including, but not limited to,pharmaceutically acceptable aqueous oral dispersions, emulsions,solutions, elixirs, gels, and syrups. See, e.g., Singh et al.,Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).In addition to the particles of egg yolk powder, the liquid dosage formsmay include additives, such as: (a) disintegrating agents; (b)dispersing agents; (c) wetting agents; (d) at least one preservative,(e) viscosity enhancing agents, (t) at least one sweetening agent, and(g) at least one flavoring agent. In some embodiments, the aqueousdispersions can further include a crystalline inhibitor.

The aqueous suspensions and dispersions described herein can remain in ahomogenous state, as defined in The USP Pharmacists' Pharmacopeia (2005edition, chapter 905), for at least 4 hours. The homogeneity should bedetermined by a sampling method consistent with regards to determininghomogeneity of the entire composition. In one embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 1 minute. In another embodiment, an aqueoussuspension can be re-suspended into a homogenous suspension by physicalagitation lasting less than 45 seconds. In yet another embodiment, anaqueous suspension can be re-suspended into a homogenous suspension byphysical agitation lasting less than 30 seconds. In still anotherembodiment, no agitation is necessary to maintain a homogeneous aqueousdispersion.

Suitable preservatives for the aqueous suspensions or dispersionsdescribed herein include, for example, potassium sorbate, parabens(e.g., methylparaben and propylparaben), benzoic acid and its salts,other esters of parahydroxybenzoic acid such as butylparaben, alcoholssuch as ethyl alcohol or benzyl alcohol, phenolic compounds such asphenol, or quaternary compounds such as benzalkonium chloride.Preservatives, as used herein, are incorporated into the dosage form ata concentration sufficient to inhibit microbial growth.

In one nonlimiting embodiment, the aqueous liquid dispersion cancomprise a sweetening agent or flavoring agent in a concentrationranging from about 0.005% to about 0.5% the volume of the aqueousdispersion. In yet another embodiment, the aqueous liquid dispersion cancomprise a sweetening agent or flavoring agent in a concentrationranging from about 0.01% to about 1.0% the volume of the aqueousdispersion.

In addition to the additives listed above, the liquid formulations canalso include inert diluents commonly used in the art, such as water orother solvents, solubilizing agents, and emulsifiers. Exemplaryemulsifiers are ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propyleneglycol,1,3-butyleneglycol, dimethylformamide, sodium lauryl sulfate, sodiumdoccusate, cholesterol, cholesterol esters, taurocholic acid,phosphotidylcholine, oils, such as cottonseed oil, groundnut oil, corngerm oil, olive oil, castor oil, and sesame oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters ofsorbitan, or mixtures of these substances, and the like.

In some embodiments, the formulations described herein can beself-emulsifying drug delivery systems (SEDDS). Emulsions aredispersions of one immiscible phase in another, usually in the form ofdroplets. Generally, emulsions are created by vigorous mechanicaldispersion.

SEDDS, as opposed to emulsions or microemulsions, spontaneously formemulsions when added to an excess of water without any externalmechanical dispersion or agitation. An advantage of SEDDS is that onlygentle mixing is required to distribute the droplets throughout thesolution. Additionally, water or the aqueous phase can be added justprior to administration, which ensures stability of an unstable orhydrophobic active ingredient. Thus, the SEDDS provides an effectivedelivery system for oral and parenteral delivery of hydrophobic activeingredients. SEDDS may provide improvements in the bioavailability ofhydrophobic active ingredients. Methods of producing self-emulsifyingdosage forms are known in the art and include, but are not limited to,for example, U.S. Pat. Nos. 5,858,401, 6,667,048, and 6,960,563.

Buccal formulations that include egg yolk powder may be administeredusing a variety of formulations known in the art. For example, suchformulations include, but are not limited to, U.S. Pat. Nos. 4,229,447,4,596,795, 4,755,386, and 5,739,136. In addition, the buccal dosageforms described herein can further include a bioerodible (hydrolysable)polymeric carrier that also serves to adhere the dosage form to thebuccal mucosa. The buccal dosage form is fabricated so as to erodegradually over a predetermined time period. Buccal drug delivery, aswill be appreciated by those skilled in the art, avoids thedisadvantages encountered with oral drug administration, e.g., slowabsorption, degradation of the active agent by fluids present in thegastrointestinal tract and/or first-pass inactivation in the liver. Withregard to the bioerodible (hydrolysable) polymeric carrier, it will beappreciated that virtually any such carrier can be used, so long as thedesired drug release profile is not compromised, and the carrier iscompatible with the egg yolk powder, and any other components that maybe present in the buccal dosage unit. Generally, the polymeric carriercomprises hydrophilic (water-soluble and water-swellable) polymers thatadhere to the wet surface of the buccal mucosa. Other components mayalso be incorporated into the buccal dosage forms described hereininclude, but are not limited to, disintegrants, diluents, binders,lubricants, flavoring, colorants, preservatives, and the like. Forbuccal or sublingual administration, the compositions may take the formof tablets, lozenges, or gels formulated in a conventional manner.

In certain embodiments, delivery systems for pharmaceutical compoundsmay be employed, such as, for example, liposomes and emulsions. Incertain embodiments, compositions provided herein can also include amucoadhesive polymer, selected from among, for example,carboxymethylcellulose, carbomer (acrylic acid polymer),poly(methylmethacrylate), polyacrylamide, polycarbophil, acrylicacid/butyl acrylate copolymer, sodium alginate and dextran.

The invention is further illustrated by the following example, whichshould not be construed as further limiting. The contents of allreferences, pending patent applications, and published patents citedthroughout this application are hereby expressly incorporated byreference.

Example

One benefit of embodiments of the present invention is the effect of eggyolk powder supplementation on the FSR of muscle proteins in men andwomen between 60 and 75 years old, or of different ages, or the like. In10 men (n=6) and women (n=4) between 60 and 75 years old, with a BMIbetween 23 and 29, ingestion of FORTETROPIN (compared with placebo) for21 days resulted in an increase in the fractional synthetic rate ofskeletal muscle proteins as compared to 10 men (n=4) and women (n=6)treated with cheese powder. Design of this FSR study is depicted inFIG. 1. As shown in FIGS. 2 through 4, the average fractional synthesisof a majority of skeletal muscle proteins in several gene ontologies wasstatistically significantly higher in the FORTETROPIN group (33/38myofibril proteins (see FIG. 2), 36/44 cytoplasmic proteins (See FIG. 3)and 15/19 mitochondrial proteins (see FIG. 4)). The overall magnitude ofincrease was 15% indicating a stimulatory effect of the egg yolk powdercomposition on muscle protein synthesis. Further, these effects wereindependent of gender or base line muscle mass of the subjects.

Subjects:

The study was comprised of 10 control subjects of 4 men and 6 women and10 FORTETROPIN subjects of 6 men and 4 women with an average age of66.35±4.45 years.

Dosing with ²H₂O:

All subjects in this study consumed approximately 100-150 ml of 70%²H₂O, divided into two to three 50 ml doses for 21 consecutive days. Onthe first day of dosing, subjects remained at a clinical site for theinitial bolus doses. During the initial four days, each subject drank150 ml of ²H₂O divided into 50 ml doses consumed at least 3 hours apart,at approximately 9:00 AM, 1:00 PM, and 5:00 PM. This is a bolus dose toincrease body water enrichment with ²H₂O rapidly to isotopic steadystate (between 1.5% and 2% enrichment). The subjects then continued toconsume 100 ml of 70% ²H₂O divided into 2 doses each day of 50 ml(morning and evening) for the remaining 17 days of methods to remain atsteady state.

D3-Creatine Dilution for Muscle Mass:

A 30 mg capsule of D3-creatine was swallowed by each subject three daysprior to initiation of FORTETROPIN treatment and the first day of ²H₂Odosing. Subjects did not need to be in the fasted condition for thisdose. Three days after the dose of D3-creatine, a morning fasted urinesample was collected. Subjects were told to void upon waking and thesecond void was collected. No food was consumed until the second urinevoid was collected. The urine sample was frozen as soon as possible.Muscle mass, and in particular an increase in muscle mass, was used as avariable in examination of the response to FORTETROPIN.

Saliva:

Saliva samples were collected to measure ²H₂O enrichment on days 4, 7,14 and 21. The enrichment of body water was used to calculate muscleprotein FSR.

Muscle Biopsy:

After 21 days of treatment with FORTETROPIN, a micro muscle biopsy wascollected on each subject midway between the patella and greatertrochanter from the vastus lateralis. About 15 mg of tissue wasobtained. Muscle sample were removed from the needle, placed in aplastic storage vial and frozen rapidly in liquid nitrogen. Samples werestored in a −70° C. freezer in accordance with exemplary embodiments.

Treatment:

Subjects were provided and asked to consume three packets of FORTETROPINpowder or placebo (isocaloric amount of cheddar cheese, 1.5 oz) at 9:00PM each evening, or the like.

Blood Samples:

Two blood samples were collected from each subject; one at baseline(study day −3) and one exactly 12 hours after the final dose ofFORTETROPIN or placebo was consumed. Blood samples were frozenimmediately and stored in a −40° C. freezer for later analysis ofcirculating myostatin levels. Height was measured on each subject andbody weight was measured weekly.

Statistical Power:

Twenty (20) evaluable subject (10 in the FORTETROPIN arm and 10 in theplacebo arm) were required to provide 80% power with a two-sided alphaof 0.05 to demonstrate the superiority of FORTETROPIN versus placebowith respect to the primary endpoint (muscle protein fractionalsynthesis rate). This estimate assumes a within group standard deviation(SD) of 0.25%/hr, with no change in FSR in the placebo group, and a0.30%/hr increase with FORTETROPIN treatment.

While the foregoing is directed to embodiments of the present invention,other and further embodiments of the invention may be devised withoutdeparting from the basic scope thereof. For example, although numerousembodiments having various features have been described herein,combinations of such various features in other combinations notdiscussed herein are contemplated within the scope of embodiments of thepresent invention.

What is claimed is:
 1. A method for inhibiting or reversing sarcopeniain a mammal, said method comprising administering to the mammal acomposition comprising egg yolk powder.
 2. The method of claim 1 whereinthe mammal is an older adult.
 3. The method of claim 1 wherein thecomposition comprises a fertilized egg yolk derived product.
 4. Themethod of claim 3 wherein the fertilized egg yolk derived product isFORTETROPIN.
 5. The method of claim 1 wherein the egg yolk powder isadministered in an amount effective to upregulate mTor pathway activity,downregulate ubiquitin proteasome pathway activity, downregulate serummyostatin levels and/or reduce ActRIIB expression.
 6. The method ofclaim 1 wherein the composition comprises avian follistatin.